Advertisement

Folia Microbiologica

, Volume 48, Issue 2, pp 168–172 | Cite as

Purification and partial characterization of α-l-arabinofuranosidase produced byThermomonospora fusca

  • M. Tuncer
  • A. S. Ball
Article

Abstract

Thermomonospora fusca produced a relatively high level of α-l-arabinofuranosidase when growing on oat spelt xylan as the main carbon and energy source. The enzyme exhibited maximum relative activity (0.136 U/g protein) at pH 9.0 with 54 and 55% activity remaining at pH of 4.5 and 11.0, respectively. The apparentKm value for the crude α-l-arabinofuranosidase preparation was 180 µmol/L 4-nitrophenyl α-l-arabinofuranoside; thevlim value was the release of 40 µmol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of ≈92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular α-l-arabinofuranosidase component. SDS-PAGE of this enzyme, developed as zymogram, did not demonstrate any activity; denaturing gel was stained and a protein band of relative molar mass of 46 kDa was revealed. Isoelectric focusing of a purified α-l-arabinofuranosidase yielded a single protein band for the corresponding activity zone with pI 7.9. The enzyme was purified approximately 21-fold the mean overall yield was about 16%.

Keywords

Fusca Single Protein Band Lignocellulose Degradation Crude Cell Lysate Native Electrophoresis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

IEF

isoelectric focusing

NPA

4-nitrophenyl α-l-arabinofuranoside

PAGE

polyacrylamide gel electrophoresis

SDS

sodium dodecyl sulfate

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bachmann S.L., McCarthy A.J.: Purification and characterization of a thermostable β-xylosidase fromThermomonospora fusca.J.Gen.Microbiol.135, 293–299 (1989).Google Scholar
  2. Bachmann S.L., McCarthy A.J.: Purification and co-operative activity of enzymes constituting the xylan-degrading system ofThermomonospora fusca.Appl.Environ.Microbiol.57, 2121–2130 (1991).PubMedGoogle Scholar
  3. Bèguin P.: Molecular biology of cellulose degradation.Ann.Rev.Microbiol.44, 219–248 (1990).CrossRefGoogle Scholar
  4. Bèguin P., Aubert J.P.: The biological degradation of cellulose.FEMS Microbiol.Rev.13, 25–58 (1994).PubMedCrossRefGoogle Scholar
  5. Betts W.B., Dart R.K., Ball A.S., Pedlar S.L.: Biosynthesis and structure of lignocellulose, pp. 139–156 in W.B. Betts (Ed.):Biodegradation Natural and Synthetic Materials. Springer-Verlag, London 1992.Google Scholar
  6. Coughlan M.P., Hazlewood G.P.: β-1,4-d-Xylan-degrading enzyme systems: biochemistry, molecular biology and applications.Biotechnol.Appl.Biochem.17, 259–289 (1993).PubMedGoogle Scholar
  7. Gilead S., Shoham Y.: Purification and characterization of α-l-arabinofuranosidase fromBacilius stearothermophilus T-6.Appl.Environ.Microbiol.61, 170–174 (1995).PubMedGoogle Scholar
  8. Goodfellow M., Williams S.T.: Ecology of actinomycetes.Ann.Rev.Microbiol.37, 189–216 (1983).CrossRefGoogle Scholar
  9. Greve L.C., Labavitch J.M., Hungate R.E.: α-l-Arabinofuranosidase fromRuminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall.Appl.Environ.Microbiol.47, 1135–1140 (1984).PubMedGoogle Scholar
  10. Iqbal M., Mercer D.K., Miller P.G.G., McCarthy A.J.: Thermostable extra-cellular peroxidases fromStreptomyces thermoviolaceus.Microbiology140, 1457–1465 (1994).CrossRefGoogle Scholar
  11. Joseleau J.P., Comtat J., Ruel K.: Chemical structure of xylans and their interaction in the plant cell walls, pp. 1–15 in J. Visser, G. Beldman, M.A. Kusters van Somerenand, A.G.J. Voragen (Eds):Xylans and Xylanases. Progress in Biotechnology, Vol. 7. Elsevier Applied Science, Amsterdam (The Netherlands) 1992Google Scholar
  12. Kaji A., Sato M., Tsutsui Y.: An α-l-arabinofuranosidase produced by the wild-typeStreptomyces sp. 17-1.Agr.Biol.Chem.45, 925–931 (1981).Google Scholar
  13. Laemmli U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature227, 680–685 (1970).PubMedCrossRefGoogle Scholar
  14. Lee S.F., Forsberg C.W.: Purification and characterization of an α-l-arabinofuranosidase fromClostridium acetobutylicum ATCC 824.Can.J.Microbiol.33, 1011–1016 (1987).CrossRefGoogle Scholar
  15. Matsuo N., Kaneko S., Kuno A., Kobayashi H., Kusakabe I.: Purification, characterization end gene cloning of two α-l-arabinofuranosidase fromStreptomyces chartreusis GS901.Biochem.J.346, 9–15 (2000).PubMedCrossRefGoogle Scholar
  16. Morales P., Madarro A., Flors A., Sendra J.M., Pérez-González J.A.: Purification and characterization of a xylanase and an arabinofuranosidase fromBacillus polymyxa.Enzyme Microbiol.Technol.17, 424–429 (1995).CrossRefGoogle Scholar
  17. Nissen A.M., Anker L., Munk N., Lange N.K.: Xylanases for the pulp and paper industry, pp. 325–337 in J. Visser, G. Beldman, M.A. Kusters van Somerenand, A.G.J. Voragen (Eds):Xylans and Xylanases. Progress in Biotechnology, Vol. 7. Elsevier Applied Science, Amsterdam (The Netherlands) 1992.Google Scholar
  18. Puls J., Poutanen K.: Mechanism of enzymatic hydrolysis of hemicelluloses (xylans) and procedures for determination of the enzyme activities involved, pp. 151–165 in M.P. Coughlan (Ed.),Enzyme Systems for Lignocellulose Degradation. Elsevier Applied Science, London 1989.Google Scholar
  19. Ramachandra M., Crawford D.L., Pometto A.L.: Extracellular enzyme activities during lignocellulose degradation byStreptomyces sp.: a comparative study of wild-type and genetically manipulated strains.Appl.Environ.Microbiol.53, 2754–2760 (1987).PubMedGoogle Scholar
  20. Reid I.D., Paice M.G.: Effects of manganese peroxidase on residual lignin of softwood kraft pulp.Appl.Environ.Microbiol.64, 2273–2274 (1998).PubMedGoogle Scholar
  21. Rob A., Ball A.S., Tuncer M., Wilson M.T.: Thermostable novel non-hem extracellular glycosylated peroxidase fromThermomonospora fusca BD25.Biotechnol.Appl.Biochem.24, 161–170 (1996).Google Scholar
  22. Schwarz W.H., Bronnenmeier K., Krause B., Lottspeich F., Staudenbauer W.L.: Debranching of arabinoxylan: properties of the thermoactive recombinant α-l-arabinofuranosidase fromClostridium stercorarium (arfB).Appl.Microbiol.Biotechnol.43, 856–860 (1995).PubMedCrossRefGoogle Scholar
  23. Senior D.J., Hamilton J., Bernier R.L. Jr.: Use ofStreptomyces lividans xylanase for bleaching of kraft pulps, pp. 555–558 in J. Visser, G. Beldman, M.A. Kusters van Somerenand, A.G.J. Voragen (Eds):Xylans and Xylanases. Progress in Biotechnology, Vol. 7. Elsevier Applied Science, Amsterdam (The Netherlands) 1992.Google Scholar
  24. Tagawa K., Kaji A.: α-l-Arabinofuranosidase fromAspergillus niger.Meth.Enzymol.160, 707–712 (1988).CrossRefGoogle Scholar
  25. Tajana E., Fiechter A., Zimmermann W.: Purification and characterization of two α-l-arabinofuranosidases fromStreptomyces diastaticus.Appl.Environ.Microbiol.58, 1447–1450 (1992).PubMedGoogle Scholar
  26. Thomson J.A.: Molecular biology of xylan degradation.FEMS Microbiol.Rev.104, 65–82 (1993).CrossRefGoogle Scholar
  27. Trigo C., Ball A.S.: Production of extracellular enzymes during the solubilization of straw byThermomonospora fasca BD25.Appl.Microbiol.Biotechnol.41, 366–372 (1994).CrossRefGoogle Scholar
  28. Tuncer M.: Characterization of β-xylosidase and α-l-arbinofuranosidase activities fromThermomonospora fusca BD25.Turk.J.Biol.24, 753–767 (2000).Google Scholar
  29. Tuncer M., Rob A., Ball A.S., Wilson M.T.: Production of extracellular lignocellulose degrading enzymes byThermomonospora fusca BD25.Biochem.Soc.Trans.24, S378 (1996).Google Scholar
  30. Tuncer M., Rob A., Ball A.S., Eady R.R., Henderson N., Wilson M.T.: Optimization of production of extracellular non-hem peroxidases byThermomonospora fusca BD25 in aerobic bio-reactor conditions.Biochem.Soc.Trans.25, S65 (1997).Google Scholar
  31. Tuncer M., Rob A., Ball A.S., Wilson M.T.: Optimization of extracellular lignocellulolytic enzyme production by a thermophilic actinomyceteThermomonospora fusca BD25.Enzyme Microbiol.Technol.25, 38–47 (1999).CrossRefGoogle Scholar
  32. Utt E.A., Eddy C.K., Keshav K.F., Ingram L.O.: Sequencing and expression of theButyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with β-d-xylosidase and α-l-arabinofuranosidase activities.Appl.Environ.Microbiol.57, 1227–1234 (1991).PubMedGoogle Scholar
  33. Viikari L., Kantelinen A., Sundquist J., Linko M.: Xylanases in bleaching: from an idea to the industry.FEMS Microbiol.Rev.13, 335–350 (1994).CrossRefGoogle Scholar
  34. Wood T.M., McCrae S.I.: Arabinoxylan-degrading enzyme system of the fungusAspergillus awamori: purification and properties of an α-l-arabinofuranosidase.Appl.Microbiol.Biotechnol.45, 538–545 (1996).PubMedGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic 2003

Authors and Affiliations

  1. 1.Biyoloji Bölümü, Fen-Edebiyat FakültesiMersin ÜniversitesiMersinTurkey
  2. 2.John Tabor Laboratories, Department of Biological SciencesUniversity of EssexColchesterUK

Personalised recommendations